The DNA mismatch repair (MMR) system is essential for maintaining the integrity of the mammalian genome and mutations in the mammalian MMR genes result in increased cancer susceptibility and meiotic failure. Eukaryotic MMR is a complex system that requires the interaction of several MutS and MutL proteins for the initiation of the repair reaction. Recent results suggest that the mammalian MMR proteins function in a number of processes which not only include the repair of base mismatches, but also the signaling of apoptosis in response to DNA damage as well as the suppression of recombination between non-identical sequences. Because it is likely that each of these activities contributes differentially to the prevention of genomic instability and cancer, it is necessary to study the impact of their individual loss. To accomplish this, we propose to create mouse lines that carry pathogenic missense mutations, which will disrupt the function of a particular motif but not completely inactivate the protein. We hypothesize that each of the distinct functions of the mitotic MutS homologs in mismatch repair, apoptosis and anti-recombination cooperate to suppress tumorigenesis. The specific aims of this proposal are: 1. To generate mouse lines that carry knock-in mutations in the ATPase domains of Msh2, Msh3 and Msh6 and analyze the resulting cancer susceptibility phenotype. This analysis will elucidate the significance of this motif in tumor suppression and also serve as a model for the generation of other Msh2, Msh3 and Msh6 knock-in alleles. 2. To analyze the effect of the Msh knock-in mutations on DNA repair functions in vitro and determine the resulting in vivo mutator phenotype in mouse tissues. 3. To study the DNA damage response in the Msh2-, Msh6- and Msh3- knock-in mouse lines. We propose to study the apoptotic response after exposure to DNA damaging agents in mouse embryonic fibroblast lines and in the in the gastrointestinal tract of the Msh knock-in mouse lines. In addition, we propose to analyze the sensitivity of embryonic stem cells and tumor cell lines derived from the knock-in mice to cisplatin and other chemotherapeutic drugs in a xenograft model in athymic nude mice. 4. To investigate the impact of the Msh2, Msh3 and Msh6 point mutations on the anti-recombination function of the MutS complexes. We will investigate DNA double strand break induced recombination in the chromosomes of embryonic stem cells to test whether the knock-in mutations result in a separation of DNA repair and anti-recombination functions in mice and assess the consequence of loss of this function on tumorigenesis.